Leptospirosis is a life-threatening zoonotic infection that causes acute renal failure and pulmonary hemorrhage. In the US, human leptospirosis is an emerging disease due to outbreaks that have occurred during disasters and sporting events and the increase in travel and recreation-related exposures. Furthermore, leptospirosis is a "neglected" disease which imparts it largest burden in populations of urban slum dwellers and subsistence farmers. More than one million cases of leptospirosis are reported each year, mostly from developing countries. Mortality from severe clinical forms of the disease is >10%. In several regions leptospirosis has emerged as the major cause of hemorrhagic fever. The critical need in addressing leptospirosis is a rapid diagnostic test that can enable clinicians to make effective decisions on therapy and management. Antimicrobial therapy, when administered early in the illness, can prevent disease progression and mortality. Timely diagnosis requires a laboratory test since the clinical presentation of early-phase leptospirosis is non-specific and often confused as being dengue or other cases of an acute febrile illness. Yet currently available tests have inadequate sensitivity (<50%) in detecting early-phase leptospirosis. Our Phase I studies have demonstrated the feasibility of developing a high-performing rapid test for leptospirosis. We have identified novel diagnostic targets, Leptospira immunoglobulin-like (Lig) proteins, which are the immunodominant antigens recognized by antibodies during infection. We have applied recombinant Lig fragments to an innovative proprietary immunoassay format, the Dual Path Platform (DPP"), and found that a DPP prototype had an overall sensitivity of 85% and specificity of 90% in evaluations of samples from leptospirosis patients from Brazil and Thailand. Furthermore, the DPP prototype had a sensitivity of 78% in identifying leptospirosis in the first 7 days of illness, the "window-of- opportunity" during which initiation of antimicrobial therapy provides greatest benefit. In this Phase II application we propose to develop and evaluate a rapid diagnostic test for leptospirosis which will have required characteristics for general use worldwide. The specific aims are to: 1) develop and optimize assay design, 2) determine diagnostic test performance in a multicenter evaluation, and 3) validate test production protocols in preparation for regulatory approval. We expect that a developed and fully validated assay will have major beneficial consequences for clinical practices worldwide by allowing physicians to make a point-of-care diagnosis and initiate timely therapeutic interventions which are required to prevent the high mortality associated with leptospirosis. PUBLIC HEALTH RELEVANCE: There is no effective prevention for human leptospirosis, a life-threatening emerging zoonotic disease, whose global burden is estimated to be as high as 500,000 cases annually. Untreated leptospirosis often progresses to multi-organ failure and/or pulmonary hemorrhage, and prompt diagnosis and early initiation of antibiotic therapy are the key intervention to prevent the morbidity and mortality associated with these complications. The research proposed here aims to develop a rapid point-of-care diagnostic test for leptospirosis which would enable antibiotic therapy to be initiated during the acute phase of the infection, when it is most effective. As part of the proposed Phase II study, we will conduct clinical evaluations of the rapid diagnostic test in preparation for regulatory approval.